CN116875532A - Environment pollutant toxicity detection method based on intestinal organoids - Google Patents
Environment pollutant toxicity detection method based on intestinal organoids Download PDFInfo
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Abstract
The application discloses an environmental pollutant toxicity detection method based on intestinal organoids, and belongs to the technical field of cell biology. The toxicity of the organoid on the environmental pollutants is detected by utilizing the intestinal organoid cultured in vitro, the organoid is suspended in a culture medium, the particle pollutants are added and fully mixed, and then the matrigel is added to find that the particle pollutants can be in direct contact with the organoid and enter the organoid, so that the contamination of the organoid particle pollutants is realized under the condition that the organoid form and activity are maintained at 2.5%, and a novel method is provided for in vitro research of the toxicity of the particle pollutants.
Description
Technical Field
The application relates to the technical field of cell biology, in particular to an environmental pollutant toxicity detection method based on intestinal organoids.
Background
With the development of society, a large amount of pollutants enter the environment, and the pollutants are transferred and amplified in a food chain, so that the pollutants are harmful to human health. Humans are exposed to ingested contaminants such as particulate contaminants, heavy metals, industrial synthetic compounds for food packaging, nanomaterials for carriers, etc. Once ingested into the human body, the intestine is the first exposed organ.
The mammalian intestine is the site of key functions such as digestion, absorption and assimilation, and is also the largest immune organ. Intestinal epithelium is composed of a number of cell types, which have different morphologies and roles. Of which the major cell types are the absorptive cells (i.e., intestinal epithelial cells and colonic cells) which are primarily responsible for the final digestion of food components and the absorption of ions, nutrients and water. These cells also have immune functions and are capable of producing pro-inflammatory cytokines and antibacterial molecules, which constitute the first line of defense against foreign material invasion. Goblet cells are the second most common cell type responsible for the production and secretion of mucins, forming mucus, with mechanical and chemoprotective effects. Secondly, there are enteroendocrine cells and the like responsible for releasing hormones that regulate intestinal function and other organ functions.
Current models for studying substance toxicity include construction of 2D cell models and animal models. However, 2D cell models are single in cell, and animal models are limited in many ways such as ethics and long time. The intestinal organoids contain critical intestinal cell types, including intestinal epithelial cells, goblet cells, enteroendocrine cells and intestinal stem cells, capable of reproducing the physiological structure of the intestinal epithelium. Actively dividing stem cell populations located within the intestinal organoids ensure long-term stable expansion and passaging of these organoids. Organoids generally have a variety of organ-specific cell types that reproduce the function of the organ represented.
The application of intestinal organoids in toxicology is mainly to study the toxicity of small molecular compounds and medicines, and because of the existence of matrigel, particulate pollutants are difficult to penetrate matrigel to enter the intestinal organoids, so that the toxicity of the particulate pollutants is difficult to study by using the intestinal organoids.
Disclosure of Invention
The application aims to provide an environmental pollutant toxicity detection method based on intestinal organoids, so as to solve the problems in the prior art.
In order to achieve the above object, the present application provides the following solutions:
the application provides a construction method of intestinal organoids, which is characterized by comprising the following steps:
(1) Taking blood vessels and fat on the surface of the intestinal tissue, and dissociating the cleaned small intestine or colorectal of the mouse;
(2) Filtering and collecting fractions, centrifuging, collecting precipitates to obtain intestinal crypts, and performing primary culture in an organoid growth medium;
(3) And (3) dissociating the cells after the primary culture to obtain the intestinal organoids.
Preferably, in step (1), the small intestine of the mouse is a small intestine section of 20cm cut at one end near the stomach; the washing is carried out by using phosphate buffer solution without calcium and magnesium at the temperature of 2-8 ℃.
Preferably, in step (1), the dissociation is performed by shearing the small intestine or colorectal of the mouse into 2mm pieces, placing the pieces in GCDR, and incubating the pieces on a shaking table at 20rpm for 20 minutes at 15-25 ℃.
Preferably, the GCDR is the mild cell dissociation reagent Stemcell 100-0485.
Preferably, in step (2), the filtration is filtration using a 70 μm sieve; the centrifugation is at 200x g for 3 minutes at 2-8 ℃.
Preferably, in step (2), the organoid growth medium is Stemcell 06005; the primary culture time is 6-10 days.
The application also provides the intestinal organoids obtained by the construction method.
The application also provides application of the intestinal organoids in detecting toxicity of environmental pollutants.
Preferably, the method for detecting toxicity of environmental pollutants comprises the following steps: re-suspending the subcultured small intestine or colorectal organoids by using the organoid growth medium, adding environmental pollutants, mixing uniformly, adding matrigel, mixing uniformly, culturing, observing the morphological changes of the small intestine or colorectal organoids, and measuring relevant physiological indexes.
Preferably, the Matrigel is 2.5% Matrigel; the environmental pollutants are particulate pollutants.
Preferably, the physiological index comprises the relevant indexes of cell viability, apoptosis and proliferation.
The application discloses the following technical effects:
the application aims to provide a method for environmental pollutant contamination by using 3D small intestine and colorectal organoids. According to the application, the organoids are suspended in a culture medium, and after the particulate pollutants are added and fully mixed, the matrigel is added to find that the particulate pollutants can be in direct contact with the organoids and enter the organoids; the organoid suspension culture method was determined by setting the concentrations of 0, 2.5%, 5%, 7.5%, 10% matrigel, and it was found that contamination of organoid particulate contaminants was achieved with organoid morphology and viability maintained at 2.5%. The method uses the characteristics of the intestinal organoids that can reproduce various cell types of the intestinal tract and the basic functions of the intestinal tract in vitro, and uses the intestinal organoids for toxicity detection of environmental pollutants. The application realizes the contamination of the particle pollutants of the intestinal organoids by suspension culture with the minimum matrigel concentration, and provides a new method for researching the toxicity of the particle pollutants in vitro.
Drawings
In order to more clearly illustrate the embodiments of the present application or the technical solutions in the prior art, the drawings that are needed in the embodiments will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments of the present application, and other drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.
FIG. 1 is a graph of the relative viability of small intestine organoids at different concentrations of PM 2.5;
FIG. 2 shows the results of RT-qPCR showing the expression levels of Ki67 at different concentrations of carbon black;
FIG. 3 shows the results of RT-qPCR showing the expression level of LGR5 mRNA at various concentrations of carbon black.
Detailed Description
Various exemplary embodiments of the application will now be described in detail, which should not be considered as limiting the application, but rather as more detailed descriptions of certain aspects, features and embodiments of the application.
It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the application. In addition, for numerical ranges in this disclosure, it is understood that each intermediate value between the upper and lower limits of the ranges is also specifically disclosed. Every smaller range between any stated value or stated range, and any other stated value or intermediate value within the stated range, is also encompassed within the application. The upper and lower limits of these smaller ranges may independently be included or excluded in the range.
Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present application. All documents mentioned in this specification are incorporated by reference for the purpose of disclosing and describing the methods and/or materials associated with the documents. In case of conflict with any incorporated document, the present specification will control.
It will be apparent to those skilled in the art that various modifications and variations can be made in the specific embodiments of the application described herein without departing from the scope or spirit of the application. Other embodiments will be apparent to those skilled in the art from consideration of the specification of the present application. The specification and examples of the present application are exemplary only.
As used herein, the terms "comprising," "including," "having," "containing," and the like are intended to be inclusive and mean an inclusion, but not limited to.
The technical scheme of the application is conventional in the field, and the reagents or raw materials are purchased from commercial sources or are disclosed.
Example 1
1. Organoid culture:
(1) Taking the small intestine or colorectal of the cleaned mouse, from which the blood vessels and fat on the surface of the intestinal tissue are removed
(2) A50 mL centrifuge tube was filled with 15mL of PBS free of calcium and magnesium, and one end of the washed intestine was gripped with forceps and suspended on the tube orifice. Starting from the bottom of the intestine, the intestine was cut into small pieces of 2mm with scissors, and these pieces were allowed to fall into the buffer inside the tube;
(3) Pre-moisten the 10mL pipette with PBS and gently blow the intestinal tract up and down three times with it;
(4) Allowing the fragments to settle by gravity, and then gently aspirating the supernatant, leaving enough liquid that the liquid surface just has gone past the tissue fragments;
(5) Adding 15mL fresh cold (2-8deg.C) PBS, repeating the pipetting step using a pre-wetted 10mL pipette, and pipetting the suspended tissue fragments up and down three times;
(6) Allowing the fragments to settle by gravity, and then gently aspirating the supernatant, leaving enough liquid that the liquid surface just has gone past the tissue fragments;
(7) Repeating the steps (7) - (8) for 15-20 times, and washing for 4-5 times after the supernatant is clear;
(8) The supernatant was removed and the tissue fragment was resuspended in 25mL room temperature (15-25 ℃) GCDR and incubated on a shaker at room temperature (15-25 ℃) for 20 minutes at 20 rpm;
(9) The tissue fragments were allowed to settle by gravity. Carefully aspirate and discard the supernatant, leaving enough liquid to clear the tissue fragments;
(10) The tissue fragments were resuspended in 10mL of cold (2-8deg.C) PBS containing 0.1% BSA and pipetted up and down three times. Standing until most of the intestinal tissue fragments settle to the bottom;
(11) Using the same pipette, carefully remove the supernatant and filter using a 70 μm filter, collect the filtrate in a clean 50mL conical tube. Discard the filter and mark filtrate as "fraction 1"; the fractions were placed on ice.
(12) Repeating steps (12) (13) to obtain fractions 2-6;
(13) The fraction was centrifuged at 290℃and x g for 5 minutes at 2-8 ℃. Carefully pour out and discard the supernatant, leave a pellet in each tube;
(14) The pellet in each tube was resuspended in 10mL of cold (2-8deg.C) PBS buffer containing 0.1% BSA. Transferring the suspension of each test tube into a clean 15mL conical tube respectively, and marking the corresponding fraction serial number;
(15) Six fractions were centrifuged at 200x g for 3 min at 2-8 ℃. The supernatant was decanted. Leaving intestinal crypt precipitate in the tube;
(16) The crypt samples were resuspended in 10mL cold (2-8deg.C) DMEM/F-12;
(17) The pipette tip was pre-wetted and 10. Mu.L of the selected fraction was removed and placed on a glass slide or cytometer. Using an inverted microscope, the number of crypts in a 10 μl sample was counted, and the calculated number of crypts was multiplied by 100, which is the number of crypts per ml in the fraction, as shown in table 1;
TABLE 1 crypt number in each fraction
| Fractions and process for preparing the fractions | Number of crypt |
| 1 | Without culturing |
| 2 | Without culturing |
| 3 | 10000 |
| 4 | 8000 |
| 5 | 5000 |
| 6 | 2500 |
(18) The number of crypts per well of a 24-well plate was about 400, the desired crypts were resuspended in 1:1 solution of medium and matrigel, 50 μl per well was added dropwise to the center of the 24-well plate, and after all the drop was completed, the plate was left to stand at 37deg.C for 30 minutes to waitCompletely solidifying;
(19) After the droplets solidify, 750 μl of room temperature organoid growth medium is gently added to each well along the well side walls using a pipette;
(20) Sterile PBS was added dropwise to the other wells to maintain the corresponding humidity;
(21) The plates were capped and incubated at 37℃and 5% CO 2 Culturing under the condition;
(22) The small intestine organoids generally grow to be mature completely in 6 days and colorectal organoids grow to be last 3 days after the small intestine organoids and colorectal organoids grow, the synapses are more, and the fluid changing time is 1 day;
(23) Typically, the small intestine organoids grow to day six, the colorectal organoids grow to day ten, and passages begin;
(24) Carefully sucking out the culture medium along the side wall of each organoid hole needing to be passaged, adding 1mL of mild cell dissociation reagent into each hole, and incubating for 1 minute at room temperature (the matrigel can be gently blown away); using a pre-wetted gun head, lightly blowing away the matrigel, and transferring the matrigel into a 50mL tube;
(25) Incubating for 15 minutes on a shaker at 20rpm, and observing the organoids under a microscope that the organoids are completely digested without large cell clusters;
(26) Centrifuging at 290Xg at 2-8deg.C for 5 min, and carefully removing supernatant;
(27) The pellet was resuspended in 10mL cold DMEM/F-12 and centrifuged at 290 Xg at 2-8deg.C for 5 min, and the supernatant discarded;
(28) Organoid and culture medium mixed solution was added at a ratio of 1:1 to resuspend organoid (matrigel may be slightly excessive due to residual DMEM/F-12 in the centrifuge tube after supernatant removal), and 50 μl of each well was inoculated in the center of 24 well plate;
(29) Placing into a 37 deg.C incubator to solidify matrigel for 30 min, gently adding culture medium along the pore wall, placing into 37 deg.C and 5% CO 2 Culturing in an incubator.
2. Contamination of particulate contaminants:
(1) Dissociation of small intestine or colorectal organoids to be passaged with mild cell dissociation reagents on a shaker for 15 min;
(2) Adding 600 mu L of organoid growth medium to resuspend organoid precipitate, and fully and uniformly mixing the precipitate;
(3) 1.2mL of culture medium containing organoids is sucked according to the number of contaminated pores, particle pollutants (the contaminated matters comprise PM2.5, 0,5, 10.20, 50 and 100 mug/mL, the contaminated matters comprise carbon black, 0, 25, 50 and 100 mug/mL, 100 mug of culture medium containing organoids is sucked by each tube, 0, 10, 20, 40, 100 and 200 mug/mL of PM2.5 solution is respectively added, for carbon black contaminated, 100 mug of culture medium containing organoids is sucked by each tube, 0, 50, 100 and 200 mug/mL of carbon black solution are respectively added, after fully mixing, 0, 2.5, 5, 7.5 and 10% Matrigel matrix is quickly added, the organoids, the particle pollutants and the Matrigel are fully mixed by blowing for twenty times (the process is carried out on ice or the process is carried out on ice for 10-20 min), the Matrigel is in a liquid state, and when the Matrigel is not added, the organoids are easy to be dissociated, and the Matrigel concentration of 2.5% and 10.5% Matrigel can be kept at the lowest, so that the Matrigel can be suspended in a state;
(4) Sucking a certain amount of organoid, particulate pollutant and matrigel mixed culture medium according to the size of the orifice plate, adding into the orifice plate, spreading the culture medium on the bottom of the plate, placing into 37 deg.C and 5% CO 2 Culturing in an incubator;
(5) Observing the change of the organoid morphology by using a microscope every six hours;
(6) Cell viability was measured 24 hours later using Cell Counting-Lite 3D Luminescent Cell ViabilityAssay;
(7) The cells are extracted, frozen sections are prepared after the cells are fixed by 4% paraformaldehyde, and related indexes such as apoptosis, proliferation and the like can be detected by using immunofluorescence staining.
The results of the detection are shown in fig. 1-3, and fig. 1 shows the effect of carbon black contamination on the viability of colorectal organoid cells, and the use of organoids at levels closer to the intestinal tract than planar cells indicates damage to the intestinal tract by the contamination. FIGS. 2 and 3 show the effect of detecting PM2.5 infection on colorectal organoid cell types such as Ki67 (cell proliferation marker), lgr5 (stem cell marker) which contain more cell types than planar cells, and are more rapid and easier than animal models.
The above embodiments are only illustrative of the preferred embodiments of the present application and are not intended to limit the scope of the present application, and various modifications and improvements made by those skilled in the art to the technical solutions of the present application should fall within the protection scope defined by the claims of the present application without departing from the design spirit of the present application.
Claims (10)
1. A method of constructing an intestinal organoid comprising the steps of:
(1) Taking blood vessels and fat on the surface of the removed intestinal tissue, and dissociating the cleaned intestinal section of the mouse;
(2) Filtering and collecting fractions, centrifuging, collecting precipitates to obtain intestinal crypts, and performing primary culture in an organoid growth medium;
(3) And (3) dissociating the cells after the primary culture to obtain the intestinal organoids.
2. The method of claim 1, wherein in step (1), the section of mouse intestine comprises the small intestine or colorectal; the small intestine is a small intestine section which is 20cm close to one end of the stomach; the washing is carried out by using phosphate buffer solution without calcium and magnesium at the temperature of 2-8 ℃.
3. The method of claim 1, wherein in step (1), the dissociation is performed by dividing the mouse intestinal tract into small segments of 2mm, placing the small segments in GCDR, and incubating the small segments on a shaker at 15-25℃for 20 minutes at 20 rpm.
4. The method of claim 1, wherein in step (2), the filtration is filtration using a 70 μm sieve; the centrifugation is at 200Xg for 3 minutes at 2-8deg.C.
5. The method of claim 1, wherein in step (2), the organoid growth medium is Stmcell06005; the primary culture time is 6-10 days.
6. An intestinal organoid obtainable by the construction method according to any one of claims 1-5.
7. Use of the intestinal organoid of claim 6 for detecting toxicity of environmental pollutants.
8. The use according to claim 7, wherein the method for detecting the toxicity of environmental pollutants comprises: re-suspending the subcultured small intestine or colorectal organoids by using the organoid growth medium, adding environmental pollutants, mixing uniformly, adding matrigel, mixing uniformly, culturing, observing the morphological changes of the small intestine or colorectal organoids, and measuring relevant physiological indexes.
9. The use according to claim 8, wherein the Matrigel is 2.5% Matrigel; the environmental pollutants are particulate pollutants.
10. The use according to claim 8, wherein the physiological index comprises an index related to cell viability, apoptosis, proliferation.
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